Post on 05-Apr-2018
Identification of active extracts of Curcuma zedoaria and their real- time cytotoxic activities
on Ovarian cancer cells and HUVEC cells
Si Lay Khaing1*, Siti Zawiah Omar1, Chung Yeng Looi2, Aditya Arya3,
Nooshin Mohebali2, Mustafa Ali Mohd4
1Department of Obstetrics &Gynaecology, Faculty of Medicine, University of Malaya,
50603 Kuala Lumpur, Malaysia
2Department of Pharmacology,Faculty of Medicine, University of Malaya, 50603 Kuala
Lumpur, Malaysia.
3School of Medicine, Taylor’s University, Lakeside Campus, 47500 Subang Jaya, Malaysia.
4Department of Pathology,Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur,
Malaysia.
*Corresponding Author:
Si Lay Khaing: Department of Obstetrics & Gynaecology, Faculty of Medicine,
University of Malaya, 50603 Kuala Lumpur, Malaysia.
Email:silay@ummc.edu.my; Fax: 6 (03) 79494193.
1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Abstract
Curcuma zedoaria(C. zedoaria) is a rare species of Curcuma. The rhizomes of C. zedoaria,
obtained from the central part of Myanmar. To our knowledge, the phytochemical components of
C. zedoaria from Myanmar and their activities on ovarian cancer cells are not reported in the
literature. The objectives of this study are to identify the active extracts of the Myanmar herbal
plant, C. zedoaria,and to determine their cytotoxicactivities on ovarian cancer cells and HUVEC
cells.Hexane, chloroform, methanol and water were used for various extractions from the
rhizome of C. zedoaria. Some of the compounds from the extracts were analysed by GC-
MS.The various extracts of C. zedoaria were tested with CaOV3(epithelial ovarian cancer cells),
SKOV3 (metastatic ovarian cancer cells), HUVEC (human umbilical vein endothelial cells)from
American Type Culture Collection (ATCC). Forcytotoxic activity, the proliferative response and
cell death of these cells were determined using Real Time Cell Analyzer (Roche) using
Paclitaxel as positive control.Hexane and chloroform extract of C. zedoaria were found to have
moderately potent cytotoxic activity on SKOV3 (metastatic ovarian cancer cells) and HUVEC
(human umbilical vein endothelial cells). Our study reveals cytotoxic activities of various
extracts of C. zedoaria from Myanmar on metastatic ovarian cancer cells and HUVEC cells.
Fifteen compounds identified in Hexane extract by using GC-MS.
Key words:
Ovarian cancer, cytotoxicity, CaOV3 cells, SKOV3 cells, HUVEC cells, C. zedoaria, Myanmar
medicinal plant
2
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Introduction
Globally, ovarian cancer is a major gynecological problem and one of the leading causes
of cancer death in women. It is the seventh most common cancer diagnosed among women in
the world. There were estimated 238719 new cases of ovarian cancer worldwide in 2012,
accounting for3.6 % of all cancers diagnosed in women [1].
Ovarian cancer cases were highest in the European region, followed by Southeast Asia
[2]. According to latest Malaysian National Cancer Registry report, ovarian cancer is fourth
commonest female cancer, accounting 6.1% of 56,713 new cases of women in 2007-2011 [3].
Sivanesaratnam[4] described as the survival rate is only 41.6% and the elusive nature of the
disease results in 70% of the cases being in an advanced stage at presentation. There are also
issues of chemo-resistance, recurrence even with second line chemotherapy, shorter disease free
survival and no adequate screening method for early detection. It affects the quality of life of the
patients. In view ofthese reasons, we aimed to test ovarian cancer cell-lines in order to assess
thepharmacological ability of selected medicinal plantsto prevent or treat ovarian cancer.
Cordell [5] has highlighted the immense potential of natural products in drug discovery
and why it is important to be involved in globalsustainable initiatives to improve health care.
Plants from the tropical rain forest are biologically and chemically diverse as they synthesize
various chemicals as a defense against pests, diseases and predators. They are an excellent
reservoir of medicines and chemical leads with which we can design and synthesize new drugs.
Curcuma is one such popular medicinal plant. C. zedoaria is a rare species which is available in
some parts of Asia and the world [6].
Curcuma zedoaria is one of the native plants of Myanmar [7]. The farms for Curcuma
are located in a particular area of central Myanmar. Cultivation starts around October and the
3
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
land yields good rhizomes. These rhizomes are usually collected in January to February every
year. The rhizomes of C.zedoaria are yellowish white, smell of camphor, and are known as
“Na-nwin Myo”. The rhizomes of C.zedoariaare different in appearance, colour and smell from
those ofC.longa/domestica.
Few reports were found on their chemical components and biological activities. Lobo et
al. [8] compiled 27 biologically active compounds isolated by various researchers. There are
some reports on their biological activities, such as antimicrobial, antifungal, anti-amoebic,
larvicidal, analgesic, anti-allergic, cytotoxic and anticancer activities. Among these
pharmacological activities, cytotoxic and anticancer activities are more interesting for cancer
prevention and new anticancer drug discovery. Determination of cytotoxicity is required for pre-
clinical screening of potential drugs, including plant extracts[9]. The cytototoxic and anti-tumour
properties of extracts and some compounds of C.zedoaria have been demonstrated on various
cancer cell lines and animal models [10-16]. Seo and co-workers [17]revealed thatthe water
extract of C.zedoaria may play an important role in the inhibition of cancer metastasis.
To date, there is a very limited number of studies in the literature on the activity of
C.zedoaria on ovarian cancer cells since the earliest work done by Syu et al in1998 [10].
Traditionally,the use of C. zedoariain pregnant women is prohibited because of the risk of
abortion, as described in the Chinese Pharmacopoeia [18]. Since medicinal herbs and their
phytochemicals are potential novel leads for developing anti-angiogenic drugs [19],we
postulated that C. zedoaria would have anti-angiogenic properties.HUVEC cell lines were used
to describe the mechanism of action of a chemotherapeutic agent related to the anti-angiogenic
properties as a classic model for anti-angiogenesis [20]. Therefore, we tested CaOv3 (epithelial
ovarian cancer cells), SKOV3 cells (metastatic ovarian cancer cells) and HUVEC(human
4
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
umbilical vein endothelial cells) in our study to investigate the cytotoxicity of various extracts of
C.zedoaria against these three cell lines.
Materials and Methods
Plant materials
The rhizomes of C. zedoaria were collected from the farm in Kyauk-Se township near Mandalay,
central Myanmar. The plant and rhizomes of C. zedoaria were authenticated by the taxonomist,
University of Traditional Medicine, Mandalay, Myanmar (UTMM).The herbarium number is
No.10 of the Herbarium Museum of UTMM.
Preparation of extracts
The extracts from rhizomes were prepared as described byArya[21] at Shimadzu-UMMC Centre
for Xenobiotics Study (SUCXes) laboratory, Pharmacology Department, Faculty of Medicine,
University of Malaya. Briefly, 500 grams of rhizomes were coarsely powdered and placed in a
cellulose thimble to extract with 100% n-hexane using hot extraction technique with a Soxhlet
extractor for 24 hr. Further extraction of the obtained defatted residue was carried out using
100% chloroform, followed by 100% (absolute) methanol and lastly with 100% pure water. The
solvents from each crude extract were dried by rotary evaporator under reduced pressureat a
maximum temperature of 40ºC.Further to the extraction, all the crude extracts were properly
stored in a freezer at -20º C until further use.
5
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
Identification and chemical analysis
All the crude extracts were subjected toGCMS-MS analysis for the qualitative analysis of
majorcompounds.The potential active compounds from various crude extracts were identified
using GCMS-MS as described by Arya et al [21].
Chemicals and consumables
CaOV3(epithelial ovarian cancer cells), SKOV3 (metastatic ovarian cancer cells) and HUVEC
(human umbilical vein endothelial cells)and McCoy’s 5A medium(modified) were purchased
from ATCC (American Type Culture Collection).Roswell Park Memorial Institute (RPMI) 1640
culture medium, penicillin-streptomycin liquid, and trypsin 2.5%(10X)were purchased from Life
Technology, USA(GIBCO). Fetal Bovine Serum, Hank’s balanced salt solution(HBSS) and
dimethyl sulfoxide(DMSO) were purchased from Sigma-Aldrich Chemicals (St. Louis,
MO,USA). The culture wares and consumables were procured from Orange Scientific(Braine-
l’Alleud, Belgium). Hexane, methanol and chloroform were supplied by Merck, Darmstadt,
Germany. E-Plate 96 and E-Plate 16 for Real Time Cell analysis (RTCA) were obtained from
Roche Diagnostics, USA.These plates were readto measure the cell proliferation using
xCELLigence Real -Time Cell Analysis (RTCA system, Roche Diagnostics GmbH, Germany).
Cell Culture
The tests were performed in the CENAR (Centre for Natural Product Research and Drug
Discovery), Department of Pharmacology and Department of Pharmacy, Faculty of Medicine,
University Malaya. CaOV3 cells(adenocarcinoma ovary, epithelial ovarian cancer cell line),
SKOV3 cells (metastatic ovarian cancer cell-line, which were derived from ascities of chemo-
6
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
resistant adenocarcinoma ovary),and HUVEC cells (human umbilical vein endothelial cell-
line)were obtained from American Type Culture Collection (ATCC). The frozen cells in cryo
vials were thawed and subcultured in a sterile manner as per protocol described by ATCC.
Initially, McCoy medium was used for CaOv3 cells and SKOV3 cell culture. Later, these cells
were gradually familiarized with RPMI 1640. Endothelial Growth Medium (EMG) was used to
maintain HUVEC cells. The cells were kept at 37oC with 5% CO2incubator. The cells in culture
flasks were observed under the microscope for growth, and were used to seed when 70-80%
confluency was achieved.
Cytotoxicity and Real-Time Cell Growth Assay
In order to measure the cell viability and cell growth in a continuous manner at multiple time
points, we performed Cytotoxicity and Real-Time Cell Growth Assay using xCELLigence Real
-Time Cell Analysis (RTCA) system, as described by Looi et al [22]. In brief, SKOV3cells in
exponential growth phase were seeded in E-96 plates with electrodes after taking background
measurement of 50ul of culture medium. The optimal cell numbers in each well were
determined before the actual experiment. After seeding the cells at a density of 3 x 103, the plate
was placed back in the RTCA system and kept with 5% CO2 at 37oC for 24 hours to grow to the
log phase. Then, the cells were treated with different concentrations of various extracts of C.
zedoaria(200, 100, 50, 25,12.5, 6.25 ug/ml) disolved in medium. Paclitaxel(taxol)
1mmol/ml(0.85 ug/ml) was used as a positive control. The cells were monitored continuously
for72 hours.
Similar procedures wereperformed to culture and treat the CaOV3 and HUVEC cells.
7
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
Results
Cytotoxicity and Cell Growth
We observed the different responses of cell growth in different types of cells to each extract of
C.zedoaria in real-time cell growth inhibition for 3 consecutive days using RTCA.These
inhibitions were in time- and dose-dependent manner (SKOV3 cell-lines in Figure 1(a,b,c,d),
CaOV3 Cell-line in Figure 2(a,b,c,d) and HUVEC cell-line in Figure 3(a,b,c,d).
The calculated IC50 doses for each extract to different cell lines were shown in Table 1. Our
results showed that water extract of C.zedoaria strongly inhibited the cell growth of SKOV3
cells (IC50= 23.50ug/ml) and moderateinhibition was noted with hexane and chloroform extract
(IC50= 60.48ug/ml,61.05ug/ml respectively). Our results also revealed that hexane and
chloroform extracts of C.zedoaria were potent growth inhibitors for HUVEC cells
(IC50=13.2ug/ml, 14.9ug/ml) and methanol and water extract were moderate growth inhibitors
for HUVEC cells (36.8ug/ml, 40.3ug/ml).
Identification of chemical components from C.zedoaria
Compounds identified in hexane fraction of C. zedoaria using GC-MS were shown in Table 2.
Discussion
Ovarian cancer is known to be a most lethal gynaecological cancer even with optimal
therapy. Therefore, the discovery of newer and more effective chemotherapeutic
agentsisinevitably required to improve the clinical outcome, survival and quality of life. In this
innovative research, we were able to evaluate the anti-proliferative effect of four different
extracts from C.zedoaria from Myanmar. We found that these extracts contained potential active
8
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
compounds according to polarity and there were more compounds detected in hexane extract of
C.zedoaria rhizomes.
Previously, some compounds were isolated from CZ from various countries such as China,
Japan, Korea, India, Bangladesh, Thailand, Indonesia and Brazil[10, 23-31].Because of the
typical smell of CZ rhizomes, we postulated that CZ might contain more chemical constituents in
hexane extract. Hexane extract contains essential oil with volatile non-polar compounds which
give rise to the aroma of a particular plant or its rhizomes. We founda majority of these
compounds in hexane extract.
The phytochemical, biological, pharmacological and ethnomedical properties of CZ were
extensively reviewed by Lobo [8]. For cytotoxic and anti-proliferative activities, some cancer
cell lines such as human lung carcinoma cell line and prostate cancer cell lines [12], lung,
nasopharyngeal cancer cells, leukaemia and murine lymphoma cells, human promyelocytic
leukemia HL-60 cells[28], liver cancer cells, melanoma cellsand HUVEC cell lines [32]were
tested in-vitrousing extracts and compounds from CZ. Regarding the ovarian cancer of our
interest, the cytotoxic activity of CZwas demonstrated against human ovarian cancer OVCAR-3
cells [10]. Recently, the compounds isolated from CZ rhizomes collected from Indonesia were
reported to exhibit strong anti-proliferative activity and also found to induce apoptotic cell death
on MCF-7 breast cancer cell line [15], and on MDA-MB-231 human breast cancer cells by
petroleum extract from CZ from China [33].
In this study, we investigated the cytotoxic activity of four crude extracts against two
ovarian cancer cell lines and primary endothelial cell lines. To the best of our knowledge, this is
the first report on the cytotoxic activity of CZ rhizomes cultivated in Myanmar.
9
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
There are various spectrophotometric or fluorometric assays for determination of cytotoxic
activities as described by Cordier and Steenkamp [9]. We chose RTCA as this technologyenables
us to measure cytotoxic effect of CZ extracts in real-time. In addition, the results can be
monitored up to 72 hours with a simultaneous comparison with a standard chemotherapeutic
drug [34]. Our findings are similar with cytotoxic activity of CZ obtained from Indonesia [14].
However, cytotoxic activities were shown against cervical, breast, colorectal and lung cancer cell
lines in their study.
We proved that our extracts, particularly hexane extract, had bioactivity on SKOV3 cells
and HUVEC cells, rather than the CAOV3 cell line. Even though water extract from CZ was
found to be highly toxic to SKOV3 cells and HUVEC cells, the compounds in water extract may
contain some fatty material in micelle forms which may contribute to the toxicity. Therefore, we
focused on hexane extract to identify compounds by using GCMS.
Conclusion
Our study showed that HE-CZ from Myanmar showed a more cytotoxic effect on Metastatic
Ovarian Cancer cells and HUVEC cells than primary ovarian cancer CaOV3 cells. This finding
demonstrates that HE-CZ from Myanmar may have potential anti-cancer activity in metastatic
ovarian cancer.
Acknowledgement
We extend our appreciation to University of Malaya for financial assistance through UMRG
grant RG-360/11HTM and UMRG-Frontier Science Grant RP021A-14AFR. Our special thanks
10
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
go to Didi Erwandi from SUCXes laboratory, Pharmacology Department, FOM, UM for his kind
technical assistance.
Conflicts of interest
The authors declare no conflict of interest.
References
1. FerlayJ,et al. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide.
IARC CancerBase No.11 [Internet] 2013. International Agency for Research on Cancer
(IARC). Available from: http://globocan.iarc.fr.
2. Stewart S.L. Ovarian Cancer Incidence: Current and Comprehensive Statistics. In
‘Ovarian Cancer- Clinical and Therapeutic Perspectives’, Samir Sarghaly(Ed). 2011.p.
1-13.
3. Azizah Ab M, Nor Saleha I. T,NoorHashimah A, Asmah Z.A, Mastulu W. Malaysian
National Cancer Registry Report 2007-2011. National Cancer Institute, Ministry of
Health, Malaysia.
4. Sivanesaratnam V, Lim B.K. Ovarian Cancer.In:Arulkumaran S, Sivanesaratnam V,
editors. Essentials of Gynaecology. 2nded. New Delhi.Jaypee Brothers Medical
Publishers; 2011. p 437-448.
5. Geoffrey A. Cordell. Natural products in drug discovery- Creating a new vision.
Phytochemistry Reviews 2002; 1: 261-273.
6. Hossain S, Kader G, Nikkon F, Yeasmin T. Cytotoxicity of the rhizome of medicinal
plants. Asian Pacific Journal of Tropical Biomedicine 2012;2(2):125-127.
11
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
7. Kress W.J, DeFilipps R.A, Farr. E, Kyi D.Y.Y. A Checklist of the Trees, Shrubs, Herbs,
and Climbers of Myanmar.Contributions from the United States National Herbarium.
2003; 45:1-590.
8. Lobo. R,Prabhu KS, Shirwaikar A. Curcuma zedoaria rocs.(white turmeric): a review of
its chemical, pharmacological and ethnomedical properties. Journal of Pharmacy and
Pharmacology 2009; 61:13-21.
9. Cordier W, Steenkamp V. Evaluation of Four Assays to Determine Cytotoxicity of
Selected Crude Medicinal Plant Extracts In vitro. British Journal of Pharmaceutical
Research 2015; 7(1): 16-21.
10. Syu W.J, Shen C.C, Don M.J, Ou J.C, Lee G.H, Sun C.M.Cytotoxicity of curcuminoids
and some novel compounds from Curcuma zedoaria. Journal of Natural Products
1998;61(12): 1531-1534.
11. Myoungae K. Cytotoxic activity of the extracts from Curcuma zedoaria. J Toxicol Public
Health 2003;19: 293–96.
12. Athima S, Arunporn I, Chawaboon D, Chatchai W, Niwat K, Pranee. C y totoxic activity
of Thai medicinal plants for cancer treatment. Songklanakarin J.Sci.Technol 2005;
27(2):469-78.
13. Lakshimi S,Padmaja G,RemaniP. Antitumour effect of Isocurcumenol isolated from
Curcuma zedoaria rhizome on human and murine cancer cells. International Journal of
Medicinal Chemistry 2011. http://dx.doi.org/10.1155/2011/253962.
14. Rahman SNSA, Wahab NA, Malek SNA. In vitro morphological assessment of apoptosis
induced by anti-proliferative constituents from rhizomes of Curcuma zedoaria. Evidence-
12
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
Based Complementary and Alternative Medicine2013.
http://dx.doi.org/10.1155/2013/257108.
15. Hamdi OAA, Rahman SNSA, Awang K,Wahab NA, Looi CY, Thomas NF, Malek SNA.
Cytotoxic constituents from the rhizomes of Curcuma zedoaria. The Scientific World
Journal 2014. Doi: 10.1155/2014/321943.
16. Hamdi OA, Anouar el H, Shilpi JA, Trabolsy ZB, Zain SB, Zakaria NS, Zulkefeli M,
Weber JF, Malek SN, Rahman SN, Awang K . A Quantum Chemical and Statistical
Study of Cytotoxic Activity of Compounds Isolated from Curcuma zedoaria.
International Journal of Molecular Sciences
2015;16:9450-9468.doi:10.3390/ijms16059450.
17. Seo WG, Hwang JC, Kang SK, Jin UH, Suh SJ, Moon SK, Kim CH (2005). Suppressive
effect of Zedoariae rhizome on pulmonary metastasis of b16 melanoma cells. Journal of
Ethnopharmacology 2005;101: 249-257.
18. Chinese Pharmacopoeia Commission 2005. Chinese Pharmacopoeia (1): 194.
19. Jeong SJ, Koh W, Lee EO, Lee HJ, Lee HJ, Bae H, Lü J, Kim SH. Antiangiogenic
Phytochemicals and Medicinal Herbs. Phytother. Research 2010;DOI: 0.1002/ptr.3224.
20. PasquierE, Carre´ M, Pourroy M, Camoin L, Rebaı¨ O, Briand C,Braguer D.
Antiangiogenic activity of paclitaxel isassociated with its cytostatic effect, mediated
by the initiation but not completion of a mitochondrial apoptotic signaling
pathway. Molecular Cancer Therapeutics 2004; 3(10):1301-1310.
21. Arya A, Achoui M, Cheah SC, Adbelwahab SI, Narrima P, Mohan S, Mustafa MR, Mohd
MA. Chloroform Fraction of Centratherum anthelminticum (L.) seed inhibits tumour
necrosis factor and exhibits pleotropic bioactivities: Inhibitory role in human tumour
13
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
cells. Evidence-Based Complementary and Alternative Medicine
2012;Doi:10.1155/2012/627256.
22. Looi CY, Arya A, Cheah FK, Muharram B, Leong KH,Mohamad K, Wong WF, Rai N,
Mustafa MR. Induction of Apoptosis in Human Breast Cancer Cells via Caspase Pathway
by Vernodalin Isolated from Centratherum anthelminticum (L.)Seeds. PLOS ONE | 2013;
http://dx.doi.org/10.1371/journal.pone.0056643.
23. Shiobara Y, Kodama M, Yasuda K, Takemoto T. Curcumenone, curcumanolide A and
curcumanolide B, three sesquiterpenoids from curcuma zedoaria. Phytochemistry 1985;
24(11): 2629-2633.
24. Shiobara Y, Kodama M, Takemoto T. Zedoarol, 13-hydroxygermacrone and curzeone,
three sesquiterpenoids from Curcuma zedoaria. Phytochemistry 1986; 25(6): 1351-1353.
25. Hong CH, Lee SK. Sesquiterpenoids from the rhizome of Curcuma zedoaria. Arch
Pharm Res 2001; 24(5): 424-426.
26. Navarro DF, Souza MM, Neto RA, Golin V, Niero R, Yunes RA, Monache FD, Filho
VC. Phytochemical analysis and analgesic properties of Curcuma zedoaria grown in
Brazil. Phytomedicine 2002; 9: 427–432.
27. Makabe H, Kuwabara A, Kamo T, Hirota M. Anti-inflammatory sesquiterpenes from
Curcuma zedoaria. Nat Prod Res. 2006; 20(7): 680-685.
28. Mau JL, Lai EYC, Wang NP, Chen CC, Chang CH, Chyau CC.Composition and
antioxidant activity of the essential oil from Curcuma zedoaria. Food Chemistry 2003;
82:583–591.
29. Park GG, Eun SH, Shim SH.Chemical constituents from Curcuma zedoaria. Biochemical
Systematics and Ecology 2012;40: 65–68.
14
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
30. Angel GR. Antioxidant and antimicrobial activity of essential oils from nine starchy
Curcuma Species. International Journal of Current Pharmaceutical Research 2012;4(2):
45-47.
31. Singh P, Singh S, Kapoor IPS, Singh G, Isidorov V, Szczepaniak L. Chemical
composition and antioxidant activities of essential oil and oleoresinsfrom Curcuma
zedoaria rhizomes, Food Bioscience 2013;74:42-48.
32. Chen W, Lu Y, Gao M, Wu J, Wang A, Shi R. Anti-angiogenesis effect of essential oil
from Curcuma zedoaria in vitro and in vivo. Journal of Ethnopharmacology
2011;133:220–226.
33. GaoXF, Li QL, Li HL, Zhang HY, Su JY, Wang B, Liu P, Zhang AQ. Extracts
from Curcuma zedoaria Inhibit Proliferation of Human Breast Cancer Cell MDA-MB-
231 In Vitro.Evidence-Based Complementary and Alternative Medicine 2014;
http://dx.doi.org/10.1155/2014/730678.
34. Atienzar FA, Tilmant K, Gerets HH, Touissant G, Speeckaert S, Hanon E, Depelchin O,
Dhalluin S. The use of real-time cell analyzer technology in drug discovery: defining
optimal cell culture conditions and assay reproducibility with different adherent cellular
models. J Biomol Screen 2011; 16(6):575–587.
15
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
Results
Table1.Real Time Cytotoxic activity of various extracts of CZ against selected human Cancer
Cell lines determinedby Real Time Cell Analyzer (RTCA)(IC50 in ug/ml)
SKOV3 CaOV3 HUVEC
Hexane 60.48 93.87 13.2
Chloroform 61.05 165.57 14.9
Methanol 75.97 106.36 36.8
Water 23.5 62.04 40.3
Figure 1 (a) Hexane extract of CZ inhibits SKOV3 cells proliferation in a time – and dose-
dependent manner ( IC50=60.48ug/ml )
16
343
344
345
346
347
348
349
350
351
352
353
Figure 1(b) Methanol extract of CZ inhibits SKOV3 cells proliferation in a time – and dose-
dependent manner ( IC50=75.97ug/ml )
Figure 1 (c) Chloroform extract of CZ inhibits SKOV3 cells proliferation in a time – and dose-
dependent manner. (IC50=61.05ug/ml )
17
354
355
356
357
358
359
360
361
362
363
Figure1 (d) Water extract of CZ inhibits SKOV3 cells proliferation in a time – and dose-
dependent manner. (IC50=23.50ug/ml )
Figure 2 (a) Hexane Extract of CZ inhibits on CaOV3 Cells proliferation in a time – and dose-
dependent manner. (IC50= 93.87 ug/ml)
18
364
365
366
367
368
369
370
371
372
373
Figure 2(b) Methanol Extract of CZ on CaOV3 Cells proliferation in a time – and dose-
dependent manner. (IC50=106.36 ug/ml)
Figure 2 (c) Chloroform Extract of CZ on CaOV3 Cells proliferation in a time – and dose-
dependent manner. (164.57 ug/ml)
19
374
375
376
377
378
379
380
381
382
383
Figure 2 (d) Water Extract of CZ on CaOV3 Cells proliferation in a time – and dose-dependent
manner. (IC50=I62.8 ug/ml)
Figure 3 (a) Hexane Extract of CZ on HUVEC Cells proliferation in a time – and dose-
dependent manner. (IC50 = 13.18ug/ml)
20
384
385
386
387
388
389
390
391
392
393
Figure 3 (b) Methanol Extract of CZ on HUVEC Cells proliferation in a time – and dose-
dependent manner. (IC50 = 36.82 ug/ml)
Figure 3 (c) Chloroform Extract of CZ on HUVEC Cells proliferation in a time – and dose-
dependent manner. (IC50 = 14.87 ug/ml)
21
394
395
396
397
398
399
400
401
402
403
Figure 3 (d) Water Extract of CZ on HUVEC Cells proliferation in a time – and dose-dependent
manner. (IC50 = 40.28 ug/ml)
22
404
405
406
407
408
409
410
411
412
Table 2.Compounds tentatively identified in Hexane fraction of C. zedoaria using GC-MS
Peak
numberRTa Mol.
weight
Mol.
formula
Similarity
IndexCompound b
1 9.653 152 C10H16O 96 L.Camphor
2 10.212 154 C10H18O 96 Isoborneol
3 10.545 154 C10H18O 96 Borneol
5 17.025 204 C15H24 89 Beta elemene
6 17.758 204 C15H24 77 Isocaryophyllen
8 19.267 204 C15H24 88 Germacrene-D
9 19.408 204 C15H24 88 Beta Selinene
10 19.483 216 C15H20O 89 Benzofuran
11 19.667 204 C15H24 85 L Selinene
12 21.133 204 C15H24 91 Germacrene-B
15 22.767 222 C15H26O 71 Epi gamma Eudesmol
16 23.208 222 C15H26O 79 Veridiflorol
19 24.05 218 C15H22O 87 Germacrone
22 26.092 232 C15H20O2 76 Iso-Velleral
26 27.800 250 C15H22O3 65 HydroxyValereric acid
RTa : retention time in minute
Compoundb :Compounds listed in order of their relative area percentage
23
413
414
415
416
417
418